Gene encoding the rat dopamine D4 receptor

ABSTRACT

A gene, flanking 5′ and 3′ sequences and derived cDNA encoding a rat D 4  dopamine receptor that is predominantly located in the cardiovascular and retinal systems is disclosed. The cDNA has been expressed in transfected mammalian cells and demonstrated to preferentially bind dopamine antagonists such as clozapine. The cDNA is useful as a probe for related D 4  dopamine receptors. Expressed in appropriate cell lines, it is useful as an in vitro screen for drugs which specifically bind to the receptor. Drugs that specifically bind to the receptor are then screened using standard methodology in rats, mice or dogs, for the physiological effects. Amino acids deduced from the determination of cDNA can be used to generate either polyclonal or monoclonal antibodies which recognize the D 4  receptor sequence but do not recognize D 1 , D 2 , D 3  or D 5  dopamenergic receptors, for use in immunocytochemical studies, and for identification and isolation via flow sorting of D4 expressing cell types. Antibodies could also be used to block or modify the effects of D4 agonists and/or antagonists. It is also demonstrated that selective stimulation or inhibition of some dopamine receptors, including D 4 , can be used to induce changes in the morphology of cells such as neurons.

This is a continuation of application Ser. No. 08/014,013, filed on Jan.28, 1993, now abandonded.

The United States government has rights in this invention by virtue of agrant from the NIMH, grant number MH45019, to Richard D. Todd, principalinvestigator.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of dopamine receptors,and is specifically a gene encoding a dopamine D₄ receptor, its flanking5′ and 3′ sequences, and its derived cDNA, and methods of use thereof inscreening for compounds having selective effects on thecardiovasculature and retinal tissues through interactions with thedopamine D₄ receptor.

Dopamine is an important neurotransmitter in the central nervous system(CNS), where it is thought to be involved in a variety of functionsincluding motor coordination, reproductive regulation, and generation ofemotions. A distinct peripheral dopaminergic system is thought to exist,although it is less well characterized. CNS dopamine receptors havehistorically been divided into two major classes, D₁ and D₂, which canbe distinguished by pharmacological, functional, and physicalcharacteristics (Kebabian and Calne, (1979) “Multiple receptors fordopamine” Nature 277:93-96; Hamblin et al., (1984) “Interactions ofagonists with D₂ dopamine receptors: evidence for a single receptorpopulation existing in multiple agonist affinity-states in rat striatalmembranes” Biochem. Pharmacol. 33:877-887; Seeman et al., (1985)“Conversion of dopamine receptors from high to low affinity fordopamine” Biochem. Pharmacol. 34:151-154; Niznik, (1987) “Dopaminereceptors: molecular structure and function” Mol. Cell. Endocrinol.54:1-22). Peripheral dopamine receptors have been divided into DA1 andDA2 subgroups, which share some but not all pharmacologicalcharacteristics with their CNS counterparts (Goldberg and Kohli, (1987)“Identification and characterization of dopamine receptors in thecardiovascular system” Cardiologia 32:1603-1607; Kohli et al., (1989)“Dopamine receptors in the stellate ganglion of the dog” Eur. J.Pharmacol. 164:265-272; Brodde, (1990) “Physiology and pharmacology ofcardiovascular catecholamine receptors; implications for treatment ofchronic heart failure” Am. Heart J. 120:1565-1572).

Molecular cloning techniques have revealed a diversity of CNS receptorsubtypes in each class. All are members of the G protein-coupledreceptor gene superfamily and have seven potential transmembrane (Tm)spanning domains. In contrast to most members of the G-protein coupledreceptor gene family, the D₂-like genes have multiple exons separated byintrons both in the coding and non-coding regions. Further diversity isgenerated by alternative splicing.

Prototypic D₂ ligand binding and signal transduction characteristicshave been found for D₂ (Bunzow et al., (1988) “Cloning and expression ofa rat D₂ dopamine receptor cDNA” Nature 336:783-787) and D₃ (Sokoloff etal., (1990) “Molecular cloning and characterization of a novel dopaminereceptor (D₃) as a target for neuroleptics” Nature 347:146-151)receptors. The recently reported human D₄ receptor also has a D₂-likepharmacological profile (Van Tol et al., (1991) “Cloning of the gene fora human dopamine D₄-receptor with high-affinity for the antipsychoticclozapine” Nature 350-610-614). Two distinct D₁ receptors have also beencloned, called D₁ (Sunahara et al., (1990) “Human dopamine D₁ receptorencoded by an intronless gene on chromosome 5” Nature 347:80-83; Zhou etal., (1990) “Cloning and expression of human and rat D₁ dopaminereceptors” Nature 347:76-80; Monsma et al., (1990) “Molecular cloningand expression of a D₁ dopamine receptor linked to adenylyl cyclaseactivation” Proc. Natl. Acad. Sci. USA 87:6723-6727; Dearry et al.,(1990) “Molecular cloning and expression of the gene for a human D₁dopamine receptor” Nature 347:72-76) and D₅ (Sunahara et al., (1991)“Cloning of the gene for a human dopamine D₅ receptor with higheraffinity for dopamine than D¹ ” Nature 350:614-619). To date noperipheral dopamine receptor has been cloned, although it has beensuggested that there is a low level of expression of D₃ in kidney(Sokoloff et al., 1990).

Van Tol et al. (1991) reported the isolation of a human D₄ receptor witha high affinity for the neuroleptic drug clozapine. Multiple variants ofthis dopamine receptor were also reported by Van Tol, et al., (1992)Nature 358, 149-154. These receptors were also the subject of PCT WO92/10571 by State of Oregon. Although the function of these particularreceptors was not identified, they are assumed to be important inbinding drugs having anti-psychotic activity.

It is an object of the present invention to provide the gene, itsflanking 5′ and 3′ sequences and the derived cDNA encoding anotherdopamine D₄ receptor present in rat cells.

It is a further object of the present invention to provide methods forexpression and screening of compounds binding the new dopamine D₄receptor.

It is another object of the present invention to provide a method forscreening for compounds having cardiovascular activity and effects onretinal tissue which specifically bind to dopamine D₄ receptors.

It is still another object of the present invention to provide a meansand method for modulation of the morphology of cells expressing D₄receptors, and other dopamine receptors, by stimulation or inhibition ofthe receptors via exposure of the cells to specific compounds.

SUMMARY OF THE INVENTION

A gene, its 5′ and 3′ flanking sequences and the derived cDNA encoding arat D₄ dopamine receptor that is predominantly located in thecardiovascular and retinal systems is disclosed. The gene has beenexpressed in transfected mammalian cells and demonstrated topreferentially bind dopamine antagonists such as clozapine.

The gene and/or cDNA is useful as a probe for related D₄ dopaminereceptors. Expressed in appropriate cell lines, it is useful as an invitro screen for drugs which specifically bind to the receptor. Drugsthat specifically bind to the receptor are then screened using standardmethodology in rats, mice or dogs, for the physiological effects.Antibodies to the protein are useful in immunocyto chemical studies,identification and isolation via flow sorting of D4 expressing celltypes, and in blocking or modifying the effects of D4 agonists and/orantagonists.

Stimulation or inhibition of the D₄ receptor, D₂ receptor, or D₃receptor, either in cells naturally expressing the receptor or whichhave been transfected with cDNAs or genes encoding anyone or more ofseveral dopamine receptors, has been demonstrated to allow modificationof the cell morphology. In one example, the number and extent ofbranching of neurites in cells transfected with dopamine receptors isincreased significantly by exposure to compounds selectively binding tothe receptors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the sequence structure of the rat dopamine D₄receptor gene. Coding regions are shown in black, noncoding regions areshown as clear boxes, and relevant restriction sites are indicated.

FIG. 2 shows the amino acid alignment of rat (rD4) and human (hD4) D₄receptors.

FIG. 3 is a comparison the structure of the Human D₄ receptor gene,which originally was reported to have an additional splice site, withthe predicted structure of the rat D₄ gene. Oligonucleotides used forreverse transcription/PCR amplification are indicated. (B) Sequence ofthe cDNA generated by PCR amplification of reverse-transcribed ratatrial mRNA as described in the text. Identity and direction of thesequencing primers are indicated. Double lines to the right of thesequence indicate regions of identity with the human D₄ receptorsequence; thick lines demarcate transmembrane domains; the single linedenotes non-identity of sequences between rat and human genes within thethird cytoplasmic loop. The analogous location of the human exon 3/exon4splice site is marked with an asterisk. The Tm VI splice site, which isconserved in all D₂-like receptors isolated to date, is indicated by anarrowhead.

FIG. 4 is a pharmacological analysis of transfected rat D₂₄₄₄ and D₄receptors and human D₃ receptors in CCL1.3 cells. FIG. 4A is graph of[³H]-spiperone (%) versus log [eticlopride, M]. FIG. 4B is a graph of[³H]-spiperone (%) versus log [(+)butaclamol, M]. FIG. 4C is a graph of[³H]-spiperone (%) versus-log [clozapine, M]. The rat D₄ gene, the ratD₂₄₄₄ cDNA and the human D₃ cDNA were inserted into the expressionvector pcDNA/neo and transfected into CCL1.3 cells. Subclones expressingthe mRNAs were expanded in medium containing G418 and assayed forcompetition of 1 nM [³H]spiperone binding by eticlopride, (+)butaclamaland clozapine, as described in Materials and Methods. Open circles,D₄-expressing cells; closed circles, D₂₄₄₄-expressing cells, triangles,D₃-expressing cells. Results are shown as mean±SD of percent ofspecifically bound [³H]spiperone remaining for an experiment done intriplicate. The apparent K_(i)'s for the D₄, D₂₄₄₄ and D₃ receptors,respectively, were: 27.0±2.9 nM, 0.067±0.21 nM and 0.16±0.02 nM, foreticlopride, 51.3±17.5 nM, 0.69±0.15 nM; and 11.2±0.8 nM for(+)butaclamal; 41.7±6.1 nM, 142.5±4.3, and 620±51.6 nM for clozapine(mean±SEM for these experiments).

FIGS. 5A, B, C and D, are bar graphs of neurite number (FIG. 5A), branchnumber (FIG. 5B), primary neurite length (FIG. 5C), and total neuriteextent (FIG. 5D), in percent change following quinpirole stimulation forcontrol MN9D cells, and D2₄₄₄, D₃, and D₄ transfected cells. The data ofthree separate experiments for each of the four cell types are expressedas the average percent change±SEM from control (unstimulated) cells.Cell numbers for the individual experiments were 100 for each conditionexcept for one experiment each for the D3 and D4 receptor expressingcells, in which there were 59 and 38 cells for each conditionrespectively. The cells were plated at low density and cultured for 90to 95 hours with or without 1 to 2 μM quinpirole. Significant changesare indicated by filled bars. Open bars represent non-significantchanges.

FIG. 6 is a comparison of control and quinpirole treated cells. Cellsfrom E15 rat mesencephalon were plated at high density, incubated for 12hours, and cultured for 90 hours in the presence (quinpirole) or absence(control) of 2 μM quinpirole. Cultures were then fixed and dopaminesynthesizing cells identified by tyrosine hydroxylase immunoreactivity.Figure shows camera lucida drawings of tyrosine hydroxylase expressingcells in confluent cultures. There are five TH positive cells in thecontrol condition and three TH positive cells in the quinpirolecondition. In the presence of quinpirole TH positive cells were larger,have more branch points and much more extensive growth cone elaboration.

DETAILED DESCRIPTION OF THE INVENTION

Dopamine receptors have been implicated in a variety of neurological andneuropsychiatric disorders. The polymerase chain reaction and lowstringency library screening were used to isolate a rat genomic cloneencoding a new dopamine receptor. Sequence data and pharmacologicalanalysis reveal this clone is the rat analog of the human D₄ receptor,which exhibits a high affinity for the antipsychotic drug clozapine. ThemRNA for this receptor shows a restricted pattern of expression in thecentral nervous system. Significant levels of expression were found inthe hypothalamus, thalamus, olfactory bulb, and frontal cortex. However,20-fold higher levels of D₄ mRNA expression were observed in thecardiovascular system. High levels are also expressed in thephotoreceptor layer of the retina. Stimulation of this receptor in thedark leads to a marked decrease in the light sensitive pool of cAMP.Thus, this receptor appears to mediate dopamine function in thecardiovascular and retinal system as well as the central nervous system.

The creation of a transfected mouse fibroblast cell line that expressesa ligand-specific receptor with the pharmacological profile of a D₂subtype has been reported by Todd et al., (1989) “Cloning ofligand-specific cell lines via gene transfer: identification of a D₂dopamine receptor subtype” Proc. Natl. Acad. Sci. USA 86:10134-10138. Asone strategy in the isolation of these sequences, a rat genomic librarywas screened with Tm-specific probes derived from D₂ (Bunzow et al.,1988) and D₃ (Sokoloff et al., 1990) consensus sequences. Using thisapproach, D₁, D₂, and D₃ rat genomic clones, as well as a clone of anunknown receptor that had a high degree of structural identity with D₂and D₃ receptor genes, were identified. Sequence data andpharmacological analyses demonstrated that this was the rat equivalentof the human D₄ gene of Van Tol (1991), although significantdifferences, as shown below, exist between the human and the rat genes.The highest levels of expression of the rat analog of the human D₄“clozapine” receptor are found in the heart and the proximal aorticarch.

EXAMPLE 1 Isolation and Characterization of the Rat D₄ Gene

The isolation and characterization of the gene and cDNA encoding the ratD₄ dopamine receptor will be further understood by reference to thefollowing detailed description.

Materials and Methods

Isolation of the Rat D₄ Gene

A lambda Dash rat spleen genomic library (Stratagene) was screened forD₂-like receptor sequences with the use of radiolabeled Tm II, III, andVI/VII probes. Oligonucleotides encompassing the indicated domains werederived from consensus sequences from the rat D₂ (Bunzow et al., 1988)and D₃ genes (Sokoloff et al., 1990). Labeling of probes, hybridization,and washing were performed according to standard methodologies, forexample, as described by Sambrook et al., (1989): Molecular Cloning: ALaboratory Manual. Cold Spring Harbor, N.Y., Cold Spring HarborLaboratory; and Feinberg and Vogelstein, (1984) “A technique forradiolabeling DNA restriction endonuclease fragments to high specificactivity” Anal. Biochem. 137:266-267. Hybridization-positive clones werefurther characterized by amplification using the polymerase chainreaction (PCR) with gene-specific probes derived from coding and intronsequences of the rat D₂ gene (O'Malley et al., (1990) “Organization andexpression of the rat D2_(A) receptor gene: identification ofalternative transcripts and a variant donor splice site” Biochemistry29:1367-1371), the rat D₃ gene (Sokoloff et al., 1990), and the rat D₁gene (Monsma et al., 1990). Hybridization-positive, PCR-negative cloneswere plaque purified and further characterized. A 3.8-kb BamHI fragmentcommon to the seven clones that were Tm VI/VII-positive but notidentified as D₂, D₃, or D₁, was subcloned and sequenced using themethod of Chen and Seeburg (1985) “Supercoil sequencing: a fast andsimple method for sequencing plasma DNA” DNA 4:165-170. DNA sequenceanalysis was performed with computer programs generated byIntelligenetics. The reported sequences are available from GenBank underaccession number M84009.

The derived gene sequence for rat dopamine D₄ receptor is shown asSequence ID No. 1. The deduced amino acid sequence is shown as SequenceID No. 2.

Expression Vectors

A full-length D2_(A444) cDNA clone (2.3 kb) was isolated from a ratstriatal library and subcloned into the HindIII site of pcDNA/neo. Agenomic D₄ fragment generated by partial NarI digestion and completeBamHI digestion was made blunt ended and ligated into the EcoRV site ofpcDNA/neo. The D₄ gene in the expression vector started at nucleotide −5and stopped 336 bp 3′ of the stop codon. DNA was purified as describedby Gandelman et al., (1990) “Species and regional differences in theexpression of cell type specific elements at the human and rat tyrosinehydroxylase gene loci” J. Neurochem. 55:2149-2152, for transfection.

Cell Culture and Transfection

Mouse CCL1.3 tk⁻ fibroblasts were grown in DMEM media supplemented with10% fetal bovine serum. Cells were plated at a density of 3×10⁶cells/10−cm dish, 12 to 24 h prior to transfection. Each plate of cellswas transfected with 20 μg of plasmid DNA by CaPO₄ precipitation, usingthe method of Chen and Okayama (1987) “High efficiency transformation ofmammalian cells by plasmid DNA” Mol. Biol. 7:2745-2752. Four hours aftertransfection, cells were shocked with 20% glycerol in DMEM for 2 min,and 48 h later the cells were split and placed in fresh mediasupplemented with 400 μg/ml of G418 (Geneticin, Gibco, activeconcentration). After two weeks, G418-resistant colonies were isolatedwith micropipette tips and screened for expression of D2_(A444) or D₄mRNA by reverse transcription/PCR analysis. Subclones expressing highlevels of D2_(A444) or D₄ mRNA were expanded and further characterized.

Binding Studies

Mouse fibroblasts expressing D2_(A444) and D₄ were grown to 70%confluence and then harvested by scraping. After they had been washedtwice in PBS, the cell pellets were resuspended in distilled water andruptured by homogenization with a Brinkman Polytron, at setting 6 for 10sec. Nuclei were removed by centrifugation for 5 min at 600 g. Membraneswere pelleted by centrifugation for 25 min at 50,000 g. The pellets wereresuspended in water and frozen at −70° C. until assayed. For receptorbinding assays, samples containing 150 μg of membrane protein werealiquoted into glass test tubes. [³H]-Spiperone (1 nm) and varyingconcentrations of competing compounds were added in a final volume of 1ml and a final buffer of 1.5 Mm CaCl₂, 5 mM MgCl₂, 5 mM KCl, 120 mMNaCl, 50 mM Tris-HCl, pH 7.4 at 20° C. The tubes were incubated for 15min at 37° C., and the assays were terminated by addition of 5 ml ofice-cold 50 Mm Tris-HCl buffer (pH 6.9), collected onto glass-fiberfilters, and washed twice with the same cold buffer in a modifiedBrandel cell harvester. The radioactivity retained on the filters wascounted in a Beckman LS 1701 scintillation counter.

Oligonucleotides

Oligonucleotide primers were synthesized on an Applied Biosystemssynthesizer. The Tm VI/VII primer set included orD-403,5′-TGCTGGCTGCCCTTCTTC-3′ (Sequence ID No. 5), which is identical tosequences within Tm VI in both D₂ and D₃ genes, and orD-404,5′-GAAGCCTTGCGGAACTC-3′ (Sequence ID No. 6), which is complementary tosequences from TM VII. For tissue distribution studies total RNA wasreverse transcribed using orD₄-515, 5′-CTGTCCACGCTGATGGCG-3′ (SequenceID No. 7), which is complementary to nucleotides 366 to 383 shown inSequence ID No. 1. Second strand synthesis and further amplificationutilized orD₄-465 and orD₄-466, 5′CAGACACCGACCAACTA-3′ (Sequence ID No.8), which is identical to nucleotides 187 to 204. Additionaloligonucleotides included orD₄-474. 5′-TGACACCCTCATGGCCAT-3′ (SequenceID No. 9), which is identical to nucleotides 309 to 326; orD₄-465,5′-TTGAAGATGGAGGGGGTG-3′ (Sequence ID No. 10), which is complementary tonucleotides 342 to 359; orD₄-501, 5′-GCACACCAAGCTTCACAG-3′ (Sequence IDNo. 11), which is identical to nucleotides 657 to 674; and orD₄-506,5′-TTGAAGGGCACTGT-TGACATAGC-3′ (Sequence ID No. 12), which iscomplimentary to nucleotides 1064 to 1085. Oligonucleotides used for insitu hybridization included orD-502, 5′-ATGGTGTTGGCAGGGAAC-TCGCTC-3′(Sequence ID No. 13), which is identical with nucleotides 124 to 193,and orD-499, 5′-GAGCGAGTTCCCTGCCAACACCAT-3′ (Sequence ID No. 14), whichis complementary to the same nucleotides.

mRNA Analysis by PCR

Total RNA was isolated from various tissues using the method ofChomczynski and Sacchi (1987) “Single-step method of RNA isolation byacid guanidium thiocyanate-phenol-chloroform extraction” Anal. Biochem.162:156-159, reverse transcribed using the method of Krug and Berger(1987) “First-strand cDNA synthesis primed with oligo(dT)” MethodsEnzymol. 152:316-325, and further amplified as described by O'Malley etal. (1990). Amplification temperatures were: denaturation at 93° C. for1 min, annealing at 52° C. for 1 min, and synthesis at 72° C. for 1 minfor 30 cycles. PCR products were transferred to nylon afterelectrophoresis in 5% polyacrylamide gels. Filters were probed with anend-labeled oligonucleotide, orD₄-474, corresponding to sequences thatare internal to the amplification set. Filters were hybridized andwashed according to the manufacturer's protocol (Schleicher andSchuell). The mRNA levels were normalized for equal amounts of the 18Sfragment of ribosomal RNA by Northern blotting followed by hybridizationwith an 18S gene fragment, using the method of Chan et al., (1984) “Thenucleotide sequence of a rat 18S ribosomal ribonucleic acid gene, and aproposal for the secondary structure of 18S ribonucleic acid” J. BiolChem 259:224-230.

In Situ Hybridization

Sense and antisense D₄ oligonucleotide probes were end-labeled withdigoxigenin-derivatized dUTP according to the manufacturer's protocols(Boehringer-Mannheim). Hybridization conditions were essentially asdescribed by Springer et al., (1991) “Non-radioactive detection of nervegrowth factor receptor. (NGFR) mRNA in rat brain using in situhybridization histochemistry” J. Histochem. Cytochem. 39:231-234. Theprobe concentration was 35 ng/ml, and hybridization was overnight at 37°C. The final stringency wash was 0.5×SSC, 22° C. for 30 min.

Results

Low stringency screening of genomic libraries with Tm-specific probeswas performed in order to isolate additional members of the dopamine D₂receptor family. Because there are regions of very high identity betweenD₂ and D₃, notably within Tm domains II and III (100% amino acididentity) and TM VI and VII (70% and 87% amino acid identity,respectively), it was reasoned that DNA fragments specific for theregion encoding each transmembrane sequence might be useful inidentifying other members of the D₂-like receptor family. In addition, acomparison of the structure of the rat D₂ gene (O'Malley et al., 1990)with that of the D₃ gene (Sokoloff et al., 1990; Giros et al., (1991)“Shorter variants of the D₃ dopamine receptor produced through variouspatterns of alternative splicing. Biochem. Biophys. Res. Commun.176:1584-1592) indicated a high degree of conservation in theintron-exon boundaries of these genes. Therefore, Tm specific probeswere constructed so as to not cross these sites, so that genomic DNAcould be used as a template. Domain-specific oligonucleotides weresynthesized and DNA amplification methodology used to createdouble-stranded DNA starting with rat genomic DNA as a template.Fragments of the appropriate size were gel purified, radiolabeled, andhybridized to a rat genomic library. The number of phage screenedcorresponded to 15 rat genomes of inserted DNA.

The results of screening the same set of filters successively with theTm II, III, and VI/VII probes are shown in Table 1.

TABLE 1 Results of screening a rat genomic library with degenerateoligonucleotides encompassing transmembrane domains II, III, and VI/VII.Number of clones of each type identified by PCR* Probe D1 D2 D3 D4 TM II13 38 24 0 TM III 7 43 20 0 TM VI/VII 0 21 25 7 *The receptor types ofhybridization-positive plaques were identified with the use ofgene-specific oligonucleotides in combination with PCR.

The feasibility of the strategy is evident from the detection of D₁ andD₃ using the indicated probes. No unknown dopamine receptors weredetected with the Tm II and III probes, suggesting either thatadditional receptors have less identity in these domains or that theseprobes are too biased towards D₂/D₃ sequences. Probes to Tm domains I,IV, and V were not made, since there is only limited identity between D₂and D₃ in these regions and no conservation of splice boundaries.Instead, the library was screened with the Tm VI/II probe. The majorityof the 53 positive clones were D₂ and D₃. However, seven clones wereclearly different and were further characterized. DNA from the sevenisolates was digested with several restriction enzymes, blotted, andprobed with the TM VI/VII probe. The smallest hybridizing fragment wassubcloned into Bluescript and sequenced with the Tm VI/VII PCR primers.

Translated sequences revealed two hydrophobic domains that had 62% and64% identity to D₂ and D₃, respectively, as well as a splice site inexactly the same position as in these genes, as described by Sokoloff etal., 1990; and O'Malley et al., (1990). Specific primers were designedfrom this sequence and used in unsuccessful screens of several cDNAlibraries by the polymerase chain reaction (PCR), including librariesprepared from basal ganglia, hypothalamus, fetal brain, and pituitary.Subsequently, a battery of D₂ and D₃ primers derived from Tm domains Ithrough V against the D₄ genomic clone were tested. They all seemed tohybridize to the same 3.8-kb BamHI fragment containing Tm regions VI andVII, suggesting that the new gene was very small in comparison with D₂and D₃. Further sequence data confirmed this premise, revealing anoverall gene structure of four exons and three introns spanningapproximately 3500 bp, as shown in FIG. 1B and Sequence I.D. No. 1.

Comparison of the structural features and nucleotide sequence of thehuman D₄ receptor (Sequence ID No. 3, nucleotide sequence, and 4,deduced amino acid sequence) isolated from a human neuroblastoma cellline, as described by Van Tol and coworkers (1991), indicated that thenew receptor is the rat analog of the D₄ receptor. The pharmacologicalprofile of the human clone has confirmed its D₂-like nature andsuggested that this receptor has a very high affinity for clozapine (5-to 15-fold higher than the D₂ receptor; Bunzow et al., (1988); Van Tolet al., 1991).

In the coding regions, the rat gene (Sequence ID No 1) shares 73% aminoacid and 77% nucleic acid sequence homology with the human D₄ gene(Sequence ID No 3). In contrast, the rat and human D₂ receptors share95% amino acid and 90% nucleic acid identity (Mack et al., (1991) “Themouse dopamine D2_(A) receptor gene: sequence homology with the rat andhuman genes and expression of alternative transcripts” J. Neurochem.57:795-801). As shown in FIG. 2, there is between 89% and 96% identitywithin the transmembrane domains of these genes. Most of the differencesbetween rat and human D₄ genes occur in the third intracytoplasmic loopwhere there is only 50% amino acid identity. In the human, this regionencompasses an unusual splice junction within intron 3 of the D₄ gene:instead of a canonical GT/AG donor/acceptor site, a TC/CT is indicated,as reported by Van Tol et al., (1991). This unconventional splice siteis not observed in the rat gene. Subsequently, Van Tol et al., (1992)have modified their interpretation of the human D4 gene structure. Thehuman and rat genes are now predicted to have the same number of intronsand exons.

The strategy depicted in FIG. 3A was used in order to rule out thepresence of a small intron, less than 30 bp, with a different unusualsplice site. Oligonucleotides were chosen flanking the bona fide slicesite within Tm VI (o506) and the putative splice site within the thirdcytoplasmic loop (o501). Amplification of genomic DNA would result in a618-bp fragment when these primers are used. The proposed model of therat D₄ gene predicts a 426-bp band for the cDNA. Rat atrial RNA wasreverse transcribed with the use of primer o506, then PCR amplificationwas performed with the 501/506 primer set. A single band of 426 bp wasobtained, which was subcloned and sequenced. FIG. 3B demonstrates thepresence of the Tm VI splice site and the absence of any additionalsplice sites within this sequence. Therefore, the rat D₄ gene has fourexons encoding an open reading frame of 368 amino acids.

EXAMPLE 2 Pharmacological Confirmation that the Putative Rat D₄ GeneCodes for A D₄ Receptor

To confirm that the putative rat D₄ gene codes for a dopamine receptoranalogous to the human D₄ receptor, the rat gene was inserted into theexpression vector pcDNA/neo and transfected into the CCL1.3 fibroblastcell line, which was then screened for [³H]-spiperone binding.Transfected cells were enriched by expansion in medium containing G418.The rat D₂ cDNA and the human D₃ cDNA were expressed in the same cellline. The results of the [³H]-spiperone binding studies are shown inFIGS. 4A, 4B, and 4C.

As determined by displacement with 1 μM eticlopride, specific binding of2 nM [³H]-spiperone was about 50% of the total bound counts for allthree receptors. Similar to the results reported by Van Tol et al. forthe human receptors, the rat D₂ receptor has a higher affinity foreticlopride and (+)butaclamol while the rat D₄ receptor has a 2- to3-fold higher affinity for clozapine. The human D₃ receptor also has ahigher affinity for eticlopride and (+) butaclamol and a much loweraffinity for clozapine than the rat D₄ receptor. The relative rank orderpotency of these three compounds for the three receptors, however,demonstrates that the rat D₄ gene codes for a dopamine receptoranalogous to the human D₄ gene.

Distribution of D₄ mRNA

Total RNA (250 μg) from the indicated regions was isolated, reversetranscribed, and amplified using primers flanking the first intron. PCRproducts were separated by electrophoresis, blotted onto nylon filters,and hybridized with an oligonucleotide internal to the PCR primers.Regions tested include adrenal medulla, adrenal cortex, occipitalcortex, temporal/parietal cortex, frontal cortex, olfactory bulb, basalganglia, hippocampus, medulla, thalamus, cerebellum, and mesencephalon.One microgram of hypothalamic total RNA and 250 ng of atrial andventricle RNA were treated as described above, except that primer orD4-465 was used. This primer generates a 171-bp product.

Various CNS and peripheral tissues were examined for the presence andrelative abundance of D₄ transcripts. Total RNA was reverse transcribedwith a D₄ exon 2-specific primer, and this procedure was followed bysecond strand synthesis and DNA amplification. The predicted D₄ PCRproduct of 195 bp is detected in only a few central nervous systemregions such as olfactory bulb, frontal cortex, and hypothalamus.Surprisingly, the D₄ PCR product is at least 20-fold more abundant inheart than in the CNS but is not detectable in liver, adrenal cortex,adrenal medulla, or kidney. Within the heart, D₄ is more abundant in theatrial/large vessel region. In separate experiments, mouse retinas wereexamined for the presence of D₄ transcripts and D₄ mRNA was found to beabundantly expressed in this tissue as well (Cohen, A. I., et al.,(1992) “Photoreceptors of mouse retinas possess D₄ receptors coupled toadenylate cyclase” Proc. Natl. Acad. Sci. USA 89, 12093-12097).

To confirm and extend these results, digoxigenin-labeled oligonucleotideprobes were used for in situ hybridization histochemistry. Sense andantisense oligonucleotides were end-labeled with digoxigenin-derivitizeddUTP and hybridized to 20 μm frozen sections of heart and brain from 6-to 8-week-old male Sprague-Dawley rats. The hybridized oligonucleotideswere visualized by alkaline phosphatase-linked anti-digoxigenin antiseraand counterstained with eosin. Color development was for 16 h. Sectionsthrough the proximal aorta show intense staining of aorta. Colordevelopment was for 22 h. There was scattered hypothalamic staining inthe region of the arcuate nucleus and the ventromedial nucleus of thehypothalamus, only a few positive cells in the striatum, no positivecells in the hippocampus, and the scattered presence of positive cellsin the thalamus.

In the CNS, D₄ mRNA-positive cells were found primarily in hypothalamicareas surrounding the third ventricle. The hypothalamic distributionoverlaps, but is not restricted to, the A11, A13, and A14 groups ofhypothalamic dopaminergic cell bodies. Few positive cells were observedin the basal ganglia, hippocampus, or cortical regions. In the heart,heavily labeled cells predominated in the proximal aorta and the outflowtract of the left ventricle, with scattered positive cells throughoutthe central fibrous body. With more sensitive color developmentconditions, the predominant atrial expression of D₄ mRNA detected by PCRis evident. The distribution of staining is most consistent with theexpression of D₄ mRNA in vascular smooth muscle and cardiac myocytes. D₄mRNA was also identified in the retinal neuronal and photoreceptorlayers in mouse (Cohen, et al., 1992).

The relatively high affinity of the human D₄ receptor for theneuroleptic drug clozapine, as reported by Van Tol et al., (1991), hasgenerated interest in the possibility that this site is responsible forclozapine's novel antipsychotic effects. Clozapine also has significanttachycardia and hypotensive side effects. These have generally beenascribed to antagonist interactions at muscarinic acetylcholine receptorsites, as reported Fitton and Heel, (1990) “Clozapine. A review of itspharmacological properties, and therapeutic use in schizophrenia” Drugs40:772-747. In the periphery, however, relatively selective D₂-likeagonists, such as piribedil, can cause vasodilation, hypotension, andbradycardia, as reported by McCoy et al., (1986) “Selective antagonismof the hypotensive effects of dopamine agonists in spontaneouslyhypertensive rats” Hypertension 8:298-302. These effects appear to bedue at least in part to inhibition of sympathetic nerve activity, Hohliet al., (1989), and can be blocked by peripheral D₂-like antagonistssuch as domperidone. Within the heart, D₂-like receptor stimulation haspositive inotropic effects, Zhao et al., (1990) “Effects of dopamine D₁and dopamine D₂ receptor agonists on coronary and peripheralhemodynamics” Eur. J. Pharmacol. 190:193-202. The demonstration of highlevels of expression of D₄ mRNA in the cardiovascular system indicatesthat some of these effects may be secondary consequences of binding toperipheral D₄ Dopamine receptors and that clozapine may be a prototypicmodel for a new class of receptor-selective agents for the treatment ofcardiovascular disorders. Of particular interest is the recent mappingof the locus for a familial form of the long Q-T syndrome to thevicinity of H-ras-1 on chromosome 11p, as reported by Keating et al.,“Linkage of a cardiac-arrhythmia, the long QT syndrome, and the HarveyRas-1 gene” Science 252:704-706 (1991). This is also the location of thehuman D₄ receptor gene, as reported by Gelernter et al., “DrD4, the D₄dopamine receptor, maps to distal 11p” Am. J. Hum. Gen. 49:340 (1991).It is possible that an abnormality of the D₄ receptor may be responsiblefor this cardiac conduction disorder.

In summary, it was found that the rat D₄ receptor mRNA was expressed atlow levels in several central nervous system regions but at much higherlevels in the heart and retina. In situ hybridization studies areconsistent with a hypothalamic autoreceptor function for the D₄ receptorin the central nervous system. The major site of expression, however,was in atrial and vascular myocytes. Therefore, the D₄ receptor, unlikethe other D₂-like subtypes, may be predominantly a peripheraldopaminergic receptor. Accordingly, this receptor should be useful as aspecific receptor for dopamine antagonists such as clozapine as well asdopamine agonists. By virtue of this specificity, many of thesecompounds can be used as regulators of blood pressure and heart rate.Depending on whether such compounds are agonists or antagonists, bloodpressure may be raised or lowered and heart rate slowed or quickened. Inaddition, such compounds would increase or decrease, respectively, theefficiency of cardiac contractions (i.e., positive or negative inotropiceffects). Similarly, such agonists and antagonists would decrease orincrease light-sensitive pools of cAMP in retinal photoreceptors andeffect the functioning of the eye. Effective dosages are determinedbased on the known dosages for these compounds for treatment of otherdisorders, screening for binding to cells expressing D₄ receptors,extrapolation to treatment of specific conditions, and other techniquesknown to those skilled in the art.

EXAMPLE 3 Morphogenic Potentials of D2, D3, and D4 Receptors

As discussed above, molecular cloning studies have defined a family ofdopamine D₂-like receptors (D₂, D₃, D₄), which are the products ofseparate genes. Stimulation of dopamine D₂-like receptors in cultures offetal cortical neurons increases the extension and branching of neurites(Todd, R. D. (1992) Biol. Psych 31, 794-807). To determine which D₂-likereceptors possess morphogenic potentials, a clonal mesencephalic cellline (MN9D) was transfected with D₂, D₃, or D₄ receptor subtypes,treated with the D₂ agonist quinpirole, and changes in morphologyquantitated.

The results demonstrated that stimulation of D₂ receptors increased thenumber and branching of neurites, with little effect on neuriteextension, while stimulation of D₃ and D₄ receptors increased thebranching and extension of neurites. These effects on neuronalmorphology could be blocked by the dopamine D₂-like receptor antagonisteticlopride. These results suggest that all of the known D₂-likereceptors may have specific developmental roles in regulating neuronalmorphogenesis of dopaminergic pathways. The types of morphologicaleffects seen suggest that developmental abnormalities of stimulation ofthese receptor subtypes may result in the neuroanatomical changes foundin many neurological and psychiatric disorders such as mentalretardation syndromes, schizophrenia, affective disorders and autism.Regulation of receptor subtype stimulation by agonists or antagonistsduring pre- or postnatal life may therefore be an effective form oftreatment to prevent or reverse the development of anatomicalabnormalities and these diseases.

Methods

Transfection

Different dopamine receptor cDNAs or genes were transfected into thedopamine containing mesencephalic cell line, MN9D. MN9D is a cell lineproduced by fusion of fetal mouse mesencephalic cells with N18TG2neuroblastoma cells, described by Choi, H., et al. (1991) Brain Res.552, 67-76. The MN9D cell line is a stable immortalized clonal cell lineestablished by fusion of the neuroblastoma cell N18TG2 with embryonicmouse mesencephalic dopamine producing neurons. Some of thecharacteristics of these cells include: the synthesis and release ofdopamine; neurite formation and immunoreactivity; production of largevoltage-sensitive sodium currents generated by depolarization;sensitivity to MPTP, a dopaminergic neurotoxin, and the ability todistinguish between the presence of dopaminergic target and non-targetcells. Additionally, neither the MN9D nor the CCL1.3 cell lines havedetectable mRNA or expressed protein for any of the D₁-like or D₂-likereceptors.

The exon 6 containing form of the rat D2 receptor (D2₄₄₄) (O'Malley, etal., “Organization and Expression of the rat D_(2A) receptor gene:identification of alternative transcripts and a variant donor splicesite” Biochem. 29:1367-1371 (1990)) (Sequence ID No. 15) and the humanD3 receptor cDNA (Giros, et al., C.R. Acad. Sci. (Paris) III, 311,501-508 (1990)) (Sequence ID No. 16) were inserted into the mammalianexpression vector pcDNA/neo (Invitrogen). The entire rat D4 (Sequence IDNo. 1) receptor gene was inserted into the same vector. All threeplasmids were transfected into MN9D cells using the glycerolshock/calcium phosphate technique of Wigler, M., et al. (1979) Cell 16,777-786; and Graham, F. and van der Eb, A (1973) Virology 52, 456-467,and permanent, clonal transfectants selected by G-418 resistance andlimiting dilution. The clonal cell lines were assayed for expression ofreceptor mRNAs by reverse transcription of total cellular RNA withreceptor specific oligonucleotides, followed by DNA amplification(O'Malley, K. L., et al, 1990) (RT/PCR) and for receptor protein by[³H]-spiperone binding (Todd, R. D., et al, 1989). Each clonal cell lineexpressed only the transfected dopamine receptor mRNA and the expressedreceptor proteins displayed the predicted pharmacological differencesfor D₂, D₃, and D₄ receptors. The average number of expressed receptorsper cell for the D₂₄₄₄, D₃, and D₄ expressing cell lines were about45,000, 15,000, and 3,500 respectively.

Morphology

The parental and transfected cell lines were plated at low density (8cells/mm²) onto poly(D-)lysine coated 35 mm culture dishes (Corning).The medium was Dubecco's Modified Eagle's Media (Gibco) containing 10%fetal bovine serum. 0.05% (w/v) G-418, an antibiotic which selects forthe transfected cells, was added to the medium for selection. The cellswere cultured in a humidified incubator under 10% CO₂ with or without1-2 μM quinpirole, a non-toxic D2-like receptor agonist.

To determine whether the stimulatory effects of quinpirole on neuriteoutgrowth could be blocked if mediated by dopamine D₂-like receptors,cells were co-cultured with 2 μM quinpirole and 1 μM eticlopride, aD₂-like receptor antagonist. K_(i)s (nM) for quinpirole are 4700±82(D2₄₄₄), 1567±247 (D3), 453,3±71.0 (D4); K_(i)s(nM) for eticlopride0.029±0.004 (D2₄₄₄), 0.46±0.12 (D3) 22.3±1.9 (all values are mean±SEM oftriplicate determinations for three to five individual assays).

Based on the K_(i) values, the micromolar concentrations of bothquinpirole and eticlopride were expected to have stimulatory orinhibitory effects at each of the D₂-like receptors, respectively.

Cells were plated at low density, cultured overnight without treatment,then drugs or medium were added to the cultures. Quinpirole was addedevery 12 hours since this reagent quickly oxidizes. Eticlopride wasadded every 24 hours, and the control cultures received an equal amountof medium at the same time as the quinpirole additions. Living cellswere photographed after 90 to 115 hours in culture using phase contrastmicroscopy (Nikon Diaphot) or a digital image processing system(Image-1). Morphologies of individual cells were quantitated at 1600×using a computer-interfaced drawing system with a digitizing light pad(Bioquant) as described by Todd, 1992; Sikich, L., Hickok, J. M., andTodd, R. D. Dev. Brain Res. 56, 269-274, 1990). Cells were measuredconsecutively over two to three dishes without knowledge of thetreatment condition. Processes shorter than 5 μm were not reliablyremeasured and were excluded from morphometric analysis.

Results

Transfection and subsequent agonist stimulation of dopamine D2_(A444),D₃, and D₄ receptors in MN9D cells results in distinct changes in cellmorphology. These persist for at least seven days in culture and can beblocked by dopamine D2-like antagonists.

Transfection Alters MN9D Morphology

The MN9D parent cell line which does not express either D₁- or D₂-likereceptors, and the transfected cells lines expressing D2_(A444), D₃ andD₄ receptors, elaborate neurites in culture. Unstimulated cells can bedistinguished from one another less than 12 hours after plating and allthe cell lines continue to develop in morphologically distinct mannersfor at least two weeks in culture. As shown for a single experiment inTable 2 (minus quinpirole), D2_(A444) expressing cells tend to have moreneurites and a larger neuritic extent. D₃ expressing cells have markedincreases in neurite number, branch number and total neuritic extent,and D₄ expressing cells most closely resemble the parent MN9D cell line.

TABLE 2 Effect of Quinpirole on Cell Morphology Transfected MN9D CellsMN9D D2_(A444) D3 D4 Quinpirole − + − + − + − + Number of Cells 100 100100 100 59 59 100 100 Neurite Number 3.25 ± .21  4.25 ± .23^(#)* 3.98 ±.21 4.72 ± .23 4.45 ± .29 4.64 ± .26  3.73 ± .23 4.12 ± .23  per Cell(μm) Branch Number  .69 ± .16 .63 ± .12  .66 ± .15 2.35 ± .35^(§) 1.91 ±.35 3.51 ± .34⁺  .51 ± .17 2.10 ± .29^(†) per cell (μm) Primary Neuritic24.54 ± 2.07 22.94 ± 2.01  31.30 ± 6.21 30.09 ± 2.02 32.50 ± 5.90 54.24± 6.69^(†) 21.56 ± 1.62 59.82 ± 6.7^(‡)   Length (μm) Total Neuritic59.75 ± 5.74 63.29 ± 4.39  78.22 ± 9.62 104.48 ± 8.95*  96.10 ± 12.34157.77 ± 13.94⁺ 58.00 ± 5.43 140.53 ± 13.56^(‡) Length (μm)

The cells were cultured with or without 2 μM quinpirole for 91-93 hours,photographed, and morphologies quantitated. All measurements were madewithout knowledge of the treatment condition and are expressed asmeans±SEM. In these analyses only neurites and neurite branches longerthan 5 μM were included because of poor interrater reliability atshorter lengths. Statistical comparisons are shown for the effects ofquinpirole on each individual cell lines. Since not all morphologicalcharacteristics were normally distributed all statistical analyses werenon-parametric and are corrected for the number of comparisons.Significant differences from no added quinpirole condition (Mann-WhitneyU test): ° p<10⁻²; ¹⁰⁸ p<10⁻³; ⁺p<10⁻⁴; ^(†)p<10⁻⁵; ^(‡)<10⁻⁶. MANOVApost hoc comparisons of morphological characteristics between cell linesare shown in Table 3.

TABLE 3 Comparisons of neurite outgrowth between the transfected celllines and the MN9D parent cells. Parameter Comparison p value ControlNeurite Number MN9D/MN9D3 .008 Branch Number MN9D/MN9D3 .004 MN9D2/MN9D3.003 MN9D3/MN9D4 <10⁻³ Ouinpirole Branch Number MN9D/MN9D2 <10⁻³MN9D/MN9D3 <10⁻⁶ MN9D/MN9D4 .002 MN9D3/MN9D4 .020 Primary Neurite LengthMN9D/MN9D3 <10⁻³ (μm) MN9D/MN9D4 <10⁻⁴ MN9D2/MN9D3 .011 MN9D2/MN9D4<10⁻⁴ Total Neuritic Extent MN9D/MN9D2 .035 (μm) MN9D/MN9D3 <10⁻⁶MN9D/MN9D4 <10⁻⁵ MN9D2/MN9D4 <10⁻⁴

Quinpirole Enhances Morphological Changes in Transfected Cells

The transfected dopamine D2-like receptor expressing cell lines havedistinct morphologies. Since the parent MN9D cell line synthesizes andreleases dopamine (Choi, H. K. Won, L. A., Kontur, L. A., et al, (1991)Brain Res. 552, 67-76) these differences may be secondary toauto-stimulation of the expressed receptors by release of endogenousdopamine. To test whether further stimulation of receptors would resultin increased morphological differences, cells were cultured in thepresence of the dopamine D₂-like agonist quinpirole. This agonist waschosen for its high affinity for all three receptors and its lowtoxicity to neuronal cells.

Culture with quinpirole resulted in increased morphological differencesbetween the transfected and parent cell lines. Differences between celllines were apparent within 24 hours of exposure to quinpirole and lastedfor at least 115 hours. Table 2 shows the results of a single experimentin which MN9D parent cells and the cell lines transfected withD2_(A444), D₃ and D₄ receptors were cultured for 91 to 93 hours with andwithout 2 μM quinpirole. Compared to MN9D cells, stimulation of thetransfected cell lines with quinpirole resulted in significant increasesin the number of branches and extension of neurites (Table 3).Stimulation of D2_(A444) receptors resulted in a four fold increase inthe frequency of branching of neurites with little effect on neuriteextension (n=100 cells for each condition, significance levels given inTable 3). D₃ receptor stimulation resulted in increases in neuritebranching and neurite length (n=59 cells for each condition). D₄receptor stimulation resulted in a small increase in neurite branchingand a large increase in neurite extension (n=100 cells for eachcondition). These effects have been observed in three independentexperiments for each receptor expressing cell line, as depicted in FIGS.5A, B, C, and D. Treatment of the MN9D parent cells with 2 μM quinpiroleresulted in no increases in neuritic outgrowth with the exception of theneurite number. As shown in Table 2, in this experiment there was asmall but statistically significant increase in neurite number. However,in two other experiments, no significant differences in anymorphological parameter were found on stimulation of MN9D cells (n=100cells per condition for each experiment).

The effects of quinpirole stimulation varied with receptor subtype, ascompared to the unstimulated state of each cell line. As shown in FIGS.5A, 5B and 5C, D2_(A444) stimulation resulted in significant percentincreases in neurite number and branch number with only a small increasein neurite length. D₄ stimulation resulted in the largest percentincrease in neurite length with no effect on neurite number. D₃stimulation resulted in marked increases in branch number and neuritelength with no effect on neurite number.

In summary, though the D₃ expressing MN9D cell line had moredifferentiated neurites in the unstimulated state, the D₂₄₄₄ expressingcell line showed a larger increase in neurite length followingquinpirole stimulation. On average, as compared to untransfected MN9Dcells, the morphological effects of transfection and stimulation arethat D2_(A444) expressing MN9D cells have more branched neurites whileD₄ expressing cells have longer less branched neurites. Stimulated, D₃expressing MN9D cells have the most highly branched neurites and thelongest total neuritic extents. The effects of quinpirole stimulation ofall three receptors can be blocked by 1 μM eticlopride. Similar resultshave been found for quinpirole stimulation of primary cultures ofdopaminergic, mesencephalic neurons which express a mixture of receptorsubtypes, as shown by FIG. 6.

In conclusion, dopamine D₃-like receptors transfected into a clonalmesencephalic cell line regulate neurite outgrowth in these cells andthe D₂ receptor subtypes appear to modulate different aspects of neuriteoutgrowth. These results suggest a role for dopamine D₂-receptors inmammalian neurodevelopment and provide support for the possibility thatdopamine receptor subtypes differentially modulate neurite outgrowth invivo. The observation of similar effects on neurite outgrowth in primarymesencephalic cultures supports the relevance of these effects fornormal and abnormal brain development.

Assuming these responses occur in vivo, then fundamental changes in theanatomy and function of mesocortical and mesostriatal pathways couldoccur via abnormal receptor stimulation. These are the same brainregions where neuropathological and neuroimaging abnormalities have beenreported for disorders such as schizophrenia (Benes, F. M., Davidson, J.and Bird, E. D. (1986) Arch. Gen. Psychiatr. 43, 31-35; Jeste, D. V. andLohr, J. B. (1989) Arch. Gen. Psychiatr. 46, 1019-1024; Pfefferbaum, A.et al. (1988) Arch. Gen. Psychiatr. 45, 633-640. The results alsoindicate that developmental stimulation or inhibition of dopaminereceptor subtypes via drugs, both therapeutically and abusively, canresult in profound pre- and postnatal changes in neuronal morphology andfunction. Developmental regulation of receptor stimulation thereforeoffers a therapeutic approach to preventing or reversing neuroanatomicalchanges associated with a variety of neurological and psychiatricdiseases.

EXAMPLE 5 Use of the Rat D₄ as a Screen for Cardiovascular Drugs andOther Biologically Useful Compounds

The cDNA or gene encoding the D₄ dopamine receptor can be expressed in avariety of mammalian cell lines, including the fibroblast cell linedescribed above, or in other commercially available cell lines such asCos cells, and used to screen for compounds which bind specifically tothe D₄ receptor. This is determined by comparing binding affinities forthe various D₁, D₂, and D₃ receptors with that of the D₄ receptor, thentesting in vivo those compounds which specifically bind the receptor. Itcan also be expressed in bacterial cells, notably E. coli, as well asother eukaryotic expression system such as Baculovirus infection ofinsect cells.

Based on the discovery that the D₄ dopamine receptor is predominantlyassociated with cardiovascular and retinal tissues, a principle use forthis screen is for compounds having an effect on the cardiovasculatureand retina, either dopamine antagonists or dopamine agonists, that actas vasoregulators or have ionotropic effects and that act on retinalcyclic AMP levels. Compounds which bind either the human or the rat D₄dopamine receptor can be screened. The typical models for physiologicaltesting of these compounds are rats, mice and dogs. Measurements can bemade in intact animals, in cardiovascular and retinal tissue explants orin isolated cells.

The gene and/or cDNA can also be used to generate probes for screeningin a manner similar to those methods described above for receptors otherthan the known D₁, D₂, D₃, and D₄ dopamine receptors. Probes are createdfrom sequences generally fourteen to seventeen nucleotides in length,and can be labelled using available technology and reagents, includingradiolabels, dyes, tomography position emission labels, magneticresonance imaging labels, enzymes, and fluorescent labels. Probes can beused directly or indirectly via standard methodologies includingpolymerase chain reaction (PCR) and methodologies to generate largerfragments of the D4 receptor. Starting with either RNA (via RT/PCR) orDNA, the D4 cDNA, and parts therein, can also be used to generate RNAtranscripts if cloned into appropriate expression vectors (cRNAs).

D4 DNA fragments, oligonucleotide probes or cRNAs, could all be used incommercial kits or sold separately to measure D4 transcript levels usingstandard techniques including PCR, in situ hybridization, and RNAse orSI protection assays.

Amino acid sequences can be deduced from fragments of D4 DNA Sequence,or the entire D4 coding sequence, generated by a variety of standardtechniques for synthesis of D4 synthetic peptides, D4 fusion proteinsand/or purification of D4 proteins (or parts thereof) from in vitrotranslated proteins derived from synthetic D4 RNA or proteinpurification per se. D4 proteins, peptides, fusion proteins or fragmentsthereof could subsequently be used for antibody production usingavailable technology including injection into a wide variety of speciesincluding mice, rats, rabbits, guinea pigs, goats, etc. for theproduction of polyclonal antisera as well as injection into mice andsubsequent utilization of fusion techniques for the production ofmonoclonal antibodies.

Oligonucleotides or larger sequences derived from the D4 mRNA orcomplementary sequences or antibodies directed against the D4 receptorcould be labelled or derivatized to be used as imaging agents forpositron emission tomography (PET) or magnetic resonance imaging (MRI)of the location of D4 receptors in vivo and in vitro.

Promoter sequences associated with the rat D4 receptor (5′ flankingsequences) may be utilized to create transgenic (non-human) animals viastandard methodologies, for example, by microinjection into embryos orhomologous recombination in embryonic stem cells. Depending upon thereporter gene utilized (Lac Z, diptheria toxin, etc.) various animalmodels can be created leading to the overexpression or loss of D4receptor activity. Additionally, promoter sequences driving foreign geneproducts such as the SV40 large T antigen could be used to createimmortalized cell lines from D4-expressing cell types. Selected use ofother foreign reporter genes (e.g. cholera toxin) can be used to makemodel systems whereby central nervous system or peripheral physiologycan be modified. Finally, from the sequence information presented insequences 1 and 2, vectors can be generated for subsequent homologousrecombination experiments in which the D4 gene is inactivated or“knocked out”, allowing determination of the physiological role of theD4 gene.

Antibodies against the D4 receptor can be used for immunocytochemicallocalization, flow cytometry identification and isolation of D4 receptorexpressing cells. Antibodies can also be used to block or modify theeffects of D4 receptor agonists and antagonists both in vivo and invitro.

The present invention is further understood by reference to thefollowing nucleotide and amino acid sequences.

Sequence 1 is the nucleotide sequence for both the non-coding andprotein coding regions of the rat D4 receptor gene loci.

Sequence 2 is the derived amino acid sequence for the rat D4 receptorprotein.

Sequence 3 is the nucleotide sequence for the human D4 receptor cDNA.

Sequence 4 is the derived amino acid sequence for the human D4 receptorprotein.

Sequences 5-14 are oligonucleotide primers used in the isolation of therat D4 receptor gene.

Sequence 15 is the nucleotide sequence for the rat D2 receptor cDNA (444amino acid form).

Sequence 16 is the nucleotide sequence for the human D3 receptor gene.

Modifications and variations of the present invention will be obvious tothose skilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe following claims.

16 3907 base pairs nucleic acid single linear DNA (genomic) NO NO Rattusnorvegicus Spleen genomic DNA misc_feature 1..3907 /note= “Rat D4 Gene”misc_feature 1..820 /note= “5′ flanking sequence to end of exon 1”intron 821..2299 exon 2300..2406 intron 2407..2499 exon 2500..3071intron 3072..3263 misc_feature 3264..3907 /note= “Exon 4 and 3′ flankingsequence” misc_feature 445..447 /note= “Start codon (initiatormethionine)” misc_feature 3463..3465 /note= “Stop codon (TGA)” K. L.Harmon, S. Tang, S. Todd, R. D.O′Malley The rat dopamine D4 receptorsequence, gene structure and demonstration of expression in thecardiovascular system. New Biol. 4 1-9 1992 1 GATCCCAAGC GTTGTTCCCTATCTGCCCAT GCGTGGGTGT CGGATGAAGA GTGAGCTTGA 60 TGTGCCCGAT TGTTCAGTATTGCTGAGCCT AGAACCCTTA GAGAAAGAGA GGAGGAGCCT 120 TAGCCTGTTA CAGAACCAGAGTTGATGGTT TCCTACGTGG AAGGACCCAA ATGCAGGAGT 180 CCAATAGTTC CACCACGTCCTCCAAGGTAT CTTGGAGAGA CGCTTTTGAC AAGCAATTTA 240 GTGGCCTGTC TCCTCGACGGGATGACTGAC TCTGACGAAT CTGGTCCAGA CAACCTGCTT 300 TCCATATAGT TTTCTGGAAGCACAAACTAA CCTGGATCAG GGGAAACATC AGTCGCTGCT 360 CCACCTTTTA TGCCAGTCACTTTGCTCTTG AATTGAAGCA TTTCTCCCTC TGCCAATTTC 420 CTAGAGTGCA GAAATTCAAGCCGTGGCGGG CGGGGCGGAG CTGGACGCTG GGGGCGGGAT 480 TCCGGATAGC CCCTGACTGCAAATCCCCAG GCTCAGCGCC TTGCAGAGTC TCAGCTAGGG 540 CGCCATGGGG AACAGCAGCGCTACTGGTGA CGGTGGGCTG TTGGCCGGGC GTGGGCCAGA 600 GTCCTTAGGT ACTGGCACCGGACTTGGGGG CGCGGGCGCG GCGGCGCTGG TGGGAGGCGT 660 GCTGCTCATC GGCATGGTGTTGGCAGGGAA CTCGCTCGTT TGCGTGAGCG TGGCCTCCGA 720 GCGCATCCTG CAGACACCCACCAACTACTT CATCGTGAGC CTGGCGGCTG CCGACCTCCT 780 CCTCGCGGTG CTGGTGTTGCCTCTCTTTGT CTACTCCGAG GTGAGCCTCG GTGCACTCTT 840 TCCCTAGCTC CATGGCTCCCAGCACCCCAG CCCCAGCGCG GTTTCACGCT TAGACCCTCA 900 GCAACCTTGA GCCAGGGGGTTTGCAGGGAC GCCAGGTCTC CTCACTCCTG ACTACCCACT 960 TTCTCCCGCT CTTCAGAATCTTTCGCCTAC TTTTCCTCCG TCACCCTACC ATCCTCTGAA 1020 TCCCTTCCTA TTCTATCTAAGATCTGCCAA CCCAGAGAGG ACTCGTGTCC CTAAAACCAA 1080 GGATCACAGA AAAATGTCTTTCCTATTTAA GCCCTGGCTC CCACTGCAGA AATACTGCAC 1140 CCCCAGCCCC CGCCAAAAGCCTGCTAGACA GGCAGGCGAA TGTGCAGACA CACATCACTG 1200 CCAATGACCA CTGCCTTTTGGGAACACACA CACACACACA CACACACACA CACACACACG 1260 CCAGCCTGTG CCAGCTCACCCTAAGGAAGA AGCCCCAAAG CCCGCAGCCA CCCAGATGCC 1320 AGGAGCTTGG AGCATTGCAGGCTGCAGGCA GAGAGGGCCT GGGCAGGATA CTCAACCTGA 1380 GGGGCTTAAA GGGAGTGGGTTCGGGACCTT TTGGAGACTA TTGGAACAGA GGCACCCCAG 1440 ATCAGGTCCT TCTCTTAGGTGGGACTGCTG TGGTTACCGG CAACTCTTCA TCTGCCCGAG 1500 GTTTGGCCAC ATTAAAACTGTTTGGGATAG GGGTGAGGAC AAAGCAGCCT GGCTGGGGGA 1560 GAGTGGGAAT AGGAATGGGGACATAGTGGC CTGGACTGGG GAAAGTGGGG TGGGATGCGC 1620 GGTATTTCTA AGGAAGAGCCTGAGTTCTGG AGCAGAGCCA GTGGCCACAC TTGACCCTAG 1680 GTCCTCCCCC AAGGCACAGACGTCACTGGG ATGATTCTTA AGCTCCTAAT CGTCCCGAAT 1740 CAGTGTGAAG GGATTTGGGGATGGGTGGTT TGAGGTGGCT GCATGCTCCT TTGCCCTTGA 1800 AAACGAATAC TCCATGCTGCAGACTCACAG AGAAGCTCAT GAGGTCTTTG TACTTTTAGG 1860 ACACACTTGT CCTCAGGACAATTGTCATAT GTCCAGCAAG TGAAGAGACC TATTCAAAGC 1920 TCCACAGCAG TGACAGTTCATGCAGGCAGG ATAACGTGCG TGTTGGAAGT GGATAGGATT 1980 TGTGTTTAGG GGGTGAAGGGTCAGGCCTAA GAATGCAGGG GCTCCTCTCC CTCAGATGGT 2040 ATTATCCTCT CGGATCTTACCCGAGCTTTT CACCTAAACA AAAGACCTAA GTCAAGAGGC 2100 AGGTCTGTTT GCCCCCTCTGTCCTCAGTTT ACACTTGTCT CCAACACATG TCTCAGGCTC 2160 ACTTTGGGCT GGTACGCCCCCTCCCCTCTA ACACACAGCC CTCCACTCCC CTCAACAAGA 2220 GCGGGGGGGT CAGAAAGCCCGCCGCTGAAA GGTCAGGTCT TGTGTTTCAT TTCTGCAACC 2280 TCTTCGTGGC CAGGTCCAGGGTGGCGTGTG GCTGCTGAGC CCCCGCCTCT GTGACACCCT 2340 CATGGCCATG GACGTCATGCTGTGCACCGC CTCCATCTTC AACCTGTGCG CCATCAGCGT 2400 GGACAGGTGG GTACCCCGGACGACCCGTCT CTTCCATTCC CATCTTCCGG TCAGCTGCTC 2460 CATTCGGCGG CCTCACCACTCCTGTGCTCC TTCCTCTAGG TTTGTGGCTG TGACCGTGCC 2520 ACTGCGCTAC AACCAGCAGGGTCAGTGCCA GCTGCTGCTC ATCGCCGCCA CGTGGCTGCT 2580 GTCTGCCGCG GTGGCTGCGCCCGTCGTGTG CGGCCTCAAT GATGTGCCCG GTCGCGATCC 2640 AACCGTGTGC TGCCTGGAGGACCGCGACTA CGTGGTCTAC TCATCCATTT GTTCCTTCTT 2700 CCTGCCCTGT CCGCTCATGCTACTGCTTTA CTGGGCCACT TTCCGTGGCT TGCGGCGCTG 2760 GGAGGCAGCC CGGCACACCAAGCTTCACAG CCGCGCGCCG CGCCGACCCA GCGGCCCGGG 2820 CCCGCCGGTG TCGGACCCTACTCAGGGTCC CCTCTTCTCA GATTGTCCGC CTCCCTCACC 2880 CAGCCTCCGG ACGAGCCCCACCGTCTCCAG CAGACCAGAG TCAGACCTCT CTCAGAGCCC 2940 CTGCAGCCCC GGGTGTCTGCTCCCTGATGC AGCGCTCGCG CAACCGCCTG CGCCGTCTTC 3000 CCGCAGAAAG AGAGGCGCCAAGATCACTGG AAGGGAGCGC AAGGCGATGA GAGTCCTGCC 3060 GGTGGTAGTT GGTGGGTTTCCGCCCTGGGA CAAGAGCTGA TAGAGGGAGG GGTCCCGGGA 3120 GCCGAGGAGG GAAGGGGGAAGGGTCCAGTT TGGAAGGGTG AAAGGTGGGG GACGGGGGTT 3180 CCTGGTTGAG AGACCTCGAGTGCAGGTGTC CTGGGTGAGG GACCTTGAGT GCAGGTGTAT 3240 AGCTCACGCC GCCCACCCCCAGGCCCCTTC CTGATGTGTT GGACGCCTTT CTTCGTGGTG 3300 CACATCACAC GGGCGCTGTGTCCGGCTTGT TTCGTGTCCC CACGCCTGGT CAGTGCTGTC 3360 ACCTGGCTGG GCTATGTCAACAGTGCCCTC AACCCCATCA TCTACACCAT CTTCAATGCC 3420 GAGTTTCGAA GTGTCTTCCGCAAGACTCTT CGTCTCCGCT GCTGAAAGAA CCGCTGATGT 3480 CTTGAGGTCA AGGGGTTCCAAGCCTGTGTG CAGAGTGCGC TGGCGGCTTT CGTTCGTCTG 3540 ATTAAATGAA GTCTTTCCTAACCATTTATC AACGCTGGGG GCTGGGAAAA AGTAAGGAAA 3600 AGAGGGAGGT CTTTTGTCTGGATGATGGGC CCGGCTAACT TCTGCCTTTG AGGATGCTGC 3660 CGGTTCAGCT CCAGGAGGCAGGAGGCTTCA GAAGTCTTTG CCCTGGAGGA GTAGGGGACC 3720 GACTACATCT GCCTTAGTTTCCGCTCAACA TGAAAAATGA CCAAGTGTTC TCCTGGGAGA 3780 GGAGCTAGAG GAATTTCCTGAGGCTCCTGG GTCCCCAGGA TCCTGTCCAG GCCTTGCTCC 3840 TTGGAGAGCT AGGGAGGGAGGGCTCTTCTG TCATTGATGG GGGAGGGGAT TCCCATTTCA 3900 GAAGCTT 3907 385 aminoacids amino acid single linear protein NO NO N-terminal Rattusnorvegicus Cardiac muscle Protein 1..385 /note= “Protein encoded by cDNAfor D4 receptor” K. L. Harmon, S. Tang, L. Todd, R. D.O′Malley The ratdopamine D4 receptor sequence, gene structure and demonstration ofexpression in the cardiovascular system. New Biol. 4 1-9 1992 2 FROM 1TO 385 2 Met Gly Asn Ser Ser Ala Thr Gly Asp Gly Gly Leu Leu Ala Gly Arg1 5 10 15 Gly Pro Glu Ser Leu Gly Thr Gly Thr Gly Leu Gly Gly Ala GlyAla 20 25 30 Ala Ala Leu Val Gly Gly Val Leu Leu Ile Gly Met Val Leu AlaGly 35 40 45 Asn Ser Leu Val Cys Val Ser Val Ala Ser Glu Arg Ile Leu GlnThr 50 55 60 Pro Thr Asn Tyr Phe Ile Val Ser Leu Ala Ala Ala Asp Leu LeuLeu 65 70 75 80 Ala Val Leu Val Leu Pro Leu Phe Val Tyr Ser Glu Gly GlyVal Trp 85 90 95 Leu Leu Ser Pro Arg Leu Cys Asp Thr Leu Met Ala Met AspVal Met 100 105 110 Leu Cys Thr Ala Ser Ile Phe Asn Leu Cys Ala Ile SerVal Asp Arg 115 120 125 Phe Val Ala Val Thr Val Pro Leu Arg Tyr Asn GlnGln Gly Gln Cys 130 135 140 Gln Leu Leu Leu Ile Ala Ala Thr Trp Leu LeuSer Ala Ala Val Ala 145 150 155 160 Ala Pro Val Val Cys Gly Leu Asn AspVal Pro Gly Arg Asp Pro Thr 165 170 175 Val Cys Cys Leu Glu Asp Arg AspTyr Val Val Tyr Ser Ser Ile Cys 180 185 190 Ser Phe Phe Leu Pro Cys ProLeu Met Leu Leu Leu Tyr Trp Ala Thr 195 200 205 Phe Arg Gly Leu Arg ArgTrp Glu Ala Ala Arg His Thr Lys Leu His 210 215 220 Ser Arg Ala Pro ArgArg Pro Ser Gly Pro Gly Pro Pro Val Ser Asp 225 230 235 240 Pro Thr GlnGly Pro Leu Phe Ser Asp Cys Pro Pro Pro Ser Pro Ser 245 250 255 Leu ArgThr Ser Pro Thr Val Ser Ser Arg Pro Glu Ser Asp Leu Ser 260 265 270 GlnSer Pro Cys Ser Pro Gly Cys Leu Leu Pro Asp Ala Ala Leu Ala 275 280 285Gln Pro Pro Ala Pro Ser Ser Arg Arg Lys Arg Gly Ala Lys Ile Thr 290 295300 Gly Arg Glu Arg Lys Ala Met Arg Val Leu Pro Val Val Val Gly Pro 305310 315 320 Phe Leu Met Cys Trp Thr Pro Phe Phe Val Val His Ile Thr ArgAla 325 330 335 Leu Cys Pro Ala Cys Phe Val Ser Pro Arg Leu Val Ser AlaVal Thr 340 345 350 Trp Leu Gly Tyr Val Asn Ser Ala Leu Asn Pro Ile IleTyr Thr Ile 355 360 365 Phe Asn Ala Glu Phe Arg Ser Val Phe Arg Lys ThrLeu Arg Leu Arg 370 375 380 Cys 385 1367 base pairs nucleic acid singlelinear mRNA NO NO Homo sapiens misc_feature 1..1367 /note= “D4 DopamineReceptor cDNA” H. H. Bunzow, J. R. Guan, H. C. Sunahara, R. K. Seeman,P. Niznik, H. B. Civelli, O.Van tol Cloning of the gene for a humandopamine D4 receptor with high affinity for the antipsychotic clozapine.Nature 350 610-614 1991 3 FROM 1 TO 1367 3 CGGGGGCGGG ACCAGGGTCCGGCCGGGGCG TGCCCCCGGG GAGGGACTCC CCGGCTTGCC 60 CCCCGGCGTT GTCCGCGGTGCTCAGCGCCC GCCCGGGCGC GCCATGGGGA ACCGCAGCAC 120 CGCGGACGCG GACGGGCTGCTGGCTGGGCG CGGGCCGGCC GCGGGGGCAT CTGCGGGGGC 180 ATCTGCGGGG CTGGCTGGGCAGGGCGCGGC GGCGCTGGTG GGGGGCGTGC TGCTCATCGG 240 CGCGGTGCTC GCGGGGAACTCGCTCGTGTG CGTGAGCGTG GCCACCGAGC GCGCCCTGCA 300 GACGCCCACC AACTCCTTCATCGTGAGCCT GGCGGCCGCC GACCTCCTCC TCGCTCTCCT 360 GGTGCTGCCG CTCTTCGTCTACTCCGAGGT CCAGGGTGGC GCGTGGCTGC TGAGCCCCCG 420 CCTGTGCGAC GCCCTCATGGCCATGGACGT CATGCTGTGC ACCGCCTCCA TCTTCAACCT 480 GTGCGCCATC AGCGTGGACAGGTTCGTGGC CGTGGCCGTG CCGCTGCGCT ACAACCGGCA 540 GGGTGGGAGC CGCCGGCAGCTGCTGCTCAT CGGCGCCACG TGGCTGCTGT CCGCGGCGGT 600 GGCGGCGCCC GTACTGTGCGGCCTCAACGA CGTGCGCGGC CGCGACCCCG CCGTGTGCCG 660 CCTGGAGGAC CGCGACTACGTGGTCTACTC GTCCGTGTGC TCCTTCTTCC TACCCTGCCC 720 GCTCATGCTG CTGCTCTACTGGGCCACGTT CCGCGGCCTG CAGCGCTGGG AGGTGGCACG 780 TCGCGCCAAG CTGCACGGCCGCGCGCCCCG CCGACCCAGC GGCCCTGGCC CGCCTTCCCC 840 CACGCCACCC GCGCCCCGCCTCCCCCAGGA CCCCTGCGGC CCCGACTGTG CGCCCCCCGC 900 GCCCGGCCTC CCCCCGGACCCCTGCGGCTC CAACTGTGCT CCCCCCGACG CCGTCAGAGC 960 CGCCGCGCTC CCACCCCAGACTCCACCGCA GACCCGCAGG AGGCGGCGTG CCAAGATCAC 1020 CGGCCGGGAG CGCAAGGCCATGAGGGTCCT GCCGGTGGTG GTCGGGGCCT TCCTGCTGTG 1080 CTGGACGCCC TTCTTCGTGGTGCACATCAC GCAGGCGCTG TGTCCTGCCT GCTCCGTGCC 1140 CCCGCGGCTG GTCAGCGCCGTCACCTGGCT GGGCTACGTC AACAGCGCCC TCAACCCCGT 1200 CATCTACACT GTCTTCAACGCCGAGTTCCG CAACGTCTTC CGCAAGGCCC TGCGTGCCTG 1260 CTGCTGAGCC GGGCACCCCCGGACGCCCCC CGGCCTGATG GCCAGGCCTC AGGGACCAAG 1320 GAGATGGGGA GGGCGCTTTTGTACGTTAAT TAAACAAATT CCTTCCC 1367 387 amino acids amino acid singlelinear protein NO NO N-terminal Homo sapiens Protein 1..387 /note=“Human D4 Receptor Protein” 4 Met Gly Asn Arg Ser Thr Ala Asp Ala AspGly Leu Leu Ala Gly Arg 1 5 10 15 Gly Pro Ala Ala Gly Ala Ser Ala GlyAla Ser Ala Gly Leu Ala Gly 20 25 30 Gln Gly Ala Ala Ala Leu Val Gly GlyVal Leu Leu Ile Gly Ala Val 35 40 45 Leu Ala Gly Asn Ser Leu Val Cys ValSer Val Ala Thr Glu Arg Ala 50 55 60 Leu Gln Thr Pro Thr Asn Ser Phe IleVal Ser Leu Ala Ala Ala Asp 65 70 75 80 Leu Leu Leu Ala Leu Leu Val LeuPro Leu Phe Val Tyr Ser Glu Val 85 90 95 Gln Gly Gly Ala Trp Leu Leu SerPro Arg Leu Cys Asp Ala Leu Met 100 105 110 Ala Met Asp Val Met Leu CysThr Ala Ser Ile Phe Asn Leu Cys Ala 115 120 125 Ile Ser Val Asp Arg PheVal Ala Val Ala Val Pro Leu Arg Tyr Asn 130 135 140 Arg Gln Gly Gly SerArg Arg Gln Leu Leu Leu Ile Gly Ala Thr Trp 145 150 155 160 Leu Leu SerAla Ala Val Ala Ala Pro Val Leu Cys Gly Leu Asn Asp 165 170 175 Val ArgGly Arg Asp Pro Ala Val Cys Arg Leu Glu Asp Arg Asp Tyr 180 185 190 ValVal Tyr Ser Ser Val Cys Ser Phe Phe Leu Pro Cys Pro Leu Met 195 200 205Leu Leu Leu Tyr Trp Ala Thr Phe Arg Gly Leu Gln Arg Trp Glu Val 210 215220 Ala Arg Arg Ala Lys Leu His Gly Arg Ala Pro Arg Arg Pro Ser Gly 225230 235 240 Pro Gly Pro Pro Ser Pro Thr Pro Pro Ala Pro Arg Leu Pro GlnAsp 245 250 255 Pro Cys Gly Pro Asp Cys Ala Pro Pro Ala Pro Gly Leu ProPro Asp 260 265 270 Pro Cys Gly Ser Asn Cys Ala Pro Pro Asp Ala Val ArgAla Ala Ala 275 280 285 Leu Pro Pro Gln Thr Pro Pro Gln Thr Arg Arg ArgArg Arg Ala Lys 290 295 300 Ile Thr Gly Arg Glu Arg Lys Ala Met Arg ValLeu Pro Val Val Val 305 310 315 320 Gly Ala Phe Leu Leu Cys Trp Thr ProPhe Phe Val Val His Ile Thr 325 330 335 Gln Ala Leu Cys Pro Ala Cys SerVal Pro Pro Arg Leu Val Ser Ala 340 345 350 Val Thr Trp Leu Gly Tyr ValAsn Ser Ala Leu Asn Pro Val Ile Tyr 355 360 365 Thr Val Phe Asn Ala GluPhe Arg Asn Val Phe Arg Lys Ala Leu Arg 370 375 380 Ala Cys Cys 385 18base pairs nucleic acid single linear cDNA NO NO misc_feature 1..18/note= “Synthetic oligonucleotide primer - Tm VI/VII primer set orD-403”K. L. Harmon, S. Tang, L. Todd, R. D.O′Malley The rat dopamine D4receptor sequence, gene structure and demonstration of expression in thecardiovascular system. New Biol. 4 1-9 1992 5 FROM 1 TO 18 5 TGCTGGCTGCCCTTCTTC 18 18 base pairs nucleic acid single linear cDNA NO NOmisc_feature 1..18 /note= “Synthetic oligonucleotide primer - TM VI inboth D2 and D3 genes and or D-404.” K. L. Harmon, S. Tang, L. Todd, R.D.O′Malley The rat dopamine D4 receptor sequence, gene structure anddemonstration of expression in the cardiovascular system. New Biol. 41-9 1991 6 FROM 1 TO 18 6 GAAGCCTTTG CGGAACTC 18 18 base pairs nucleicacid single linear cDNA NO NO misc_feature 1..18 /note= “Syntheticoligonucleotide primer - reverse transcribed using orD4-515, and iscomplementary to nucleotides 366 to 383 in K. L. Harmon, S. Tang, L.Todd, R. D.O′Malley The rat dopamine D4 receptor sequence, genestructure and demonstration of expression in the cardiovascular system.New Biol. 4 1-9 1992 7 FROM 1 TO 18 7 CTGTCCACGC TGATGGCG 18 18 basepairs nucleic acid single linear cDNA NO NO misc_feature 1..18 /note=”Synthetic oligonucleotide primer - utilized orD4-465 and orD4-466 andis identical to nucleotides 187 to 204 in Sequence K. L. Harmon, S.Tang, L. Todd, R. D.O′Malley The rat dopamine D4 receptor sequence, genestructure and demonstration of expression in the cardiovascular system.New Biol. 4 1-9 1992 8 FROM 1 TO 18 8 CAGACACCGA CCAACTAC 18 18 basepairs nucleic acid single linear cDNA NO NO misc_feature 1..18 /note=“Synthetic oligonucleotide primer - included orD4-474 and is identicalto nucleotides 309 to 326 in Sequence I.D. No. 1.” K. L. Harmon, S.Tang, L. Todd, R. D.O′Malley The rat dopamine D4 receptor sequence, genestructure and demonstration of expression in the cardiovascular system.New Biol. 4 1-9 1992 9 FROM 1 TO 18 9 TGACACCCTC ATGGCCAT 18 18 basepairs nucleic acid single linear cDNA NO NO misc_feature 1..18 /note=“Synthetic oligonucleotide primer - included orD4-465 and iscomplementary to nucleotides 342 to 359 in Sequence I.D. No. 1.” K. L.Harmon, S. Tang, L. Todd, R. D.O′Malley The rat dopamine D4 receptorsequence, gene structure and demonstration of expression in thecardiovascular system. New Biol. 4 1-9 1992 10 FROM 1 TO 18 10TTGAAGATGG AGGGGGTG 18 18 base pairs nucleic acid single linear cDNA NONO misc_feature 1..18 /note= “Synthetic oligonucleotide primer -included orD4-501 and is identical to nucleotides 657 to 674 in SequenceI.D. No. 1.” K. L. Harmon, S. Tang, L. Todd, R. D.O′Malley The ratdopamine D4 receptor sequence, gene structure and demonstration ofexpression in the cardiovascular system. New Biol. 4 1-9 1992 11 FROM 1TO 18 11 GCACACCAAG CTTCACAG 18 23 base pairs nucleic acid single linearcDNA NO NO misc_feature 1..23 /note= “Synthetic oligonucleotide primer -included orD4-506 and is complementary to nucleotides 1064 to 1085 inSequence I.D. No. 1.” K. L. Harmon, S. Tang, L. Todd, R. D.O′Malley Therat dopamine D4 receptor sequence, gene structure and demonstration ofexpression in the cardiovascular system. New Biol. 4 1-9 1992 12 FROM 1TO 23 12 TTGAAGGGCA CTGTTGACAT AGC 23 24 base pairs nucleic acid singlelinear cDNA NO NO misc_feature 1..24 /note= “Synthetic oligonucleotideincluded orD-502 and is identical to nucleotides 124 to 193 in SequenceI.D. No. 1.” K. L. Harmon, S. Tang, L. Todd, R. D.O′Malley The ratdopamine D4 receptor sequence, gene structure and demonstration ofexpression in the cardiovascular system. New Biol. 4 1-9 1992 13 FROM 1TO 24 13 ATGGTGTTGG CAGGGAACTC GCTC 24 24 base pairs nucleic acid singlelinear cDNA NO NO misc_feature 1..24 /note= “Synthetic oligonucleotideincluded orD-499 and is complementary to nucleotides 124 to 193 inSequence I.D. No. 1.” K. L. Harmon, S. Tang, L. Todd, R. D.O′Malley Therat dopamine D4 receptor sequence, gene structure and demonstration ofexpression in the cardiovascular system. New Biol. 4 1-9 1992 14 FROM 1TO 24 14 GAGCGAGTTC CCTGCCAACA CCAT 24 2428 base pairs nucleic acidsingle linear cDNA NO NO Rattus norvegicus Cardiac Muscle misc_feature1..2428 /note= “Rat d2 receptor sequence” J. R. Van Tol, H. H.M. Grandy,D. K. Albert, P. Salon, J. Christie, M. Machida, C. A. Neve, K. A.Civelli, O.Bunzow Cloning and expression of a rat D2 dopamine receptorcDNA. Nature 336 783-787 1988 15 FROM 1 TO 2428 15 CCACCCAGTG GCCCCACTGCCCCAATGGAT CCACTGAACC TGTCCTGGTA CGATGACGAT 60 CTGGAGAGGC AGAACTGGAGCCGGCCCTTC AATGGGTCAG AAGGGAAGGC AGACAGGCCC 120 CACTACAACT ACTATGCCATGCTGCTCACC CTCCTCATCT TTATCATCGT CTTTGGCAAT 180 GTGCTGGTGT GCATGGCTGTATCCCGAGAG AAGGCTTTGC AGACCACCAC CAACTACTTG 240 ATAGTCAGCC TTGCTGTGGCTGATCTTCTG GTGGCCACAC TGGTAATGCC GTGGGTTGTC 300 TACCTGGAGG TGGTGGGTGAGTGGAAATTC AGCAGGATTC ACTGTGACAT CTTTGTCACT 360 CTGGATGTCA TGATGTGCACAGCAAGCATC CTGAACCTGT GTGCCATCAG CATTGACAGG 420 TACACAGCTG TGGCAATGCCCATGCTGTAT AACACACGCT ACAGCTCCAA GCGCCGAGTT 480 ACTGTCATGA TTGCCATTGTCTGGGTCCTG TCCTTCACCA TCTCCTGCCC ACTGCTCTTC 540 GGACTCAACA ATACAGACCAGAATGAGTGT ATCATTGCCA ACCCTGCCTT TGTGGTCTAC 600 TCCTCCATTG TCTCATTCTACGTGCCCTTC ATCGTCACTC TGCTGCTGTA TATCAAAATC 660 TACATCGTCC TCCGGAAGCGCCGGAAGCGG GTCAACACCA AGCGCAGCAG TCGAGCTTTC 720 AGAGCCAACC TGAAGACACCACTCAAGGGC AACTGTACCC ACCCTGAGGA CATGAAACTC 780 TGCACCGTTA TCATGAAGTCTAATGGGAGT TTCCCAGTGA ACAGGCGGAG AATGGATGCT 840 GCCCGCCGAG CTCAGGAGCTGGAAATGGAG ATGCTGTCAA GCACCAGCCC CCCAGAGAGG 900 ACCCGGTATA GCCCCATCCCTCCCAGTCAC CACCAGCTCA CTCTCCCTGA TCCATCCCAC 960 CACGGCCTAC ATAGCAACCCTGACAGTCCT GCCAAACCAG AGAAGAATGG GCACGCCAAG 1020 ATTGTCAATC CCAGGATTGCCAAGTTCTTT GAGATCCAGA CCATGCCCAA TGGCAAAACC 1080 CGGACCTCCC TTAAGACGATGAGCCGCAGA AAGCTCTCCC AGCAGAAGGA GAAGAAAGCC 1140 ACTCAGATGC TTGCCATTGTTCTCGGTGTG TTCATCATCT GCTGGCTGCC CTTCTTCATC 1200 ACGCACATCC TGAATATACACTGTGATTGC AACATCCCAC CAGTCCTCTA CAGCGCCTTC 1260 ACATGGCTGG GCTATGTCAACAGTGCCGTC AACCCCATCA TCTACACCAC CTTCAACATC 1320 GAGTTCCGCA AGGCCTTCATGAAGATCTTG CACTGCTGAG TCTGCCCCTT GCCTGCACAG 1380 CAGCTGCTTC CCACCTCCCTGCCTATGCAG GCCAGACCTC ATCCCTGCAA GCTGTGGGCA 1440 GAAAGGCCCA GATGGACTTGGCCTTCTCTC GACCCTGCAG GCCCTGCAGT GTTAGCTTGG 1500 CTCGATGCCC CTCTCTGCCCACACACCCTC ATCCTGCCAG GGTAGGGCCA GGGAGACTGG 1560 TATCTTACCA GCTCTGGGGTTGGACCCATG GCTCAGGGCA GCTCACAGAG TGCCCCTCTC 1620 ATATCCAGAC CCTGTCTCCTTGGCACCAAA GATGCAGCGG CCTTCCTTGA CCTTCCTCTT 1680 GGGCACAGAA ACTAGCTCAGTGGTCGAGCA CACCCTGATC GCTGGCTTGG CCTGGCCCTT 1740 GCTTGCCTGT GCCAGATCAGGTGGTGGGAG GGAGCAACAG TTCTTACTTT ATAGGAACCA 1800 CATAGGAAAG CAGGGAACACGCCAAGTCCT CCAGGCAACA TCAGTGTCAG GAGACACACA 1860 TAAACACCAG GTAGCTCCATGGACCCCAGA GAAACTGAGG CTGAAAAATC TGTTTTCCAC 1920 TCCAACTCTA GTGTGAGTCCCTACTTTTCA TAGCCATGGG TATTACTATG TCCTACCTTG 1980 TTATAGTATC CCATGGGGTTTCTGTACCAT TTGGGGGAAA ACAACTCTAA TCCTCAAGGG 2040 CCCCAAGAGA ATCTGTAAGGAGAAAAATAG GCTGATCTCC CTCTACTCTC CAATCCACTC 2100 CACCACTTCT TGATATACCTTGGATGTATC CATTCCTCAC AGCAAATGCT GGCCAGTCAG 2160 GCCTTGGACC AGTGTTGGAGTTGAAGCTGG ATGTGGTAAC TTGGGGCTCT TTGGGGCTGG 2220 GGGGGTTGTT AACATCGTCTCTCTTCCATA TCTCTTCCTT CCCAGTGCCT CTGCCTTAGA 2280 AGAGGCTGTG GATGGGGTGCTGGGACTGCT GATACCATTG GGCCTGGCTG AATGAGGAGG 2340 GGAAGCTGCA GTTTGGAGGGTTCTGGGATC CAACTCTGTA ACATCACTAT ACCTGCACCA 2400 AAACTAATAA AACCTTGACAAGAGTCAA 2428 1261 base pairs nucleic acid single linear cDNA NO NO Homosapiens misc_feature 1..1261 /note= “Human d3 cDNA” B. Martres, M. P.Sokoloff, P. Schwartz, J. C.Giros C.R. Acad. Sci. (Paris) III 311501-508 1990 16 FROM 1 TO 1261 16 TGGGCTATGG CATCTCTGAG TCAGCTGAGTAGCCACCTGA ACTACACCTG TGGGGCAGAG 60 AACTCCACAG GTGCCAGCCA GGCCCGCCCACATGCCTACT ATGCCCTCTC CTACTGCGCG 120 CTCATCCTGG CCATCGTCTT CGGCAATGGCCTGGTGTGCA TGGCTGTGCT GAAGGAGCGG 180 GCCCTGCAGA CTACCACCAA CTACTTAGTAGTGAGCCTGG CTGTGGCAGA CTTGCTGGTG 240 GCCACCTTGG TGATGCCCTG GGTGGTATACCTGGAGGTGA CAGGTGGAGT CTGGAATTTC 300 AGCCGCATTT GCTGTGATGT TTTTGTCACCCTGGATGTCA TGATGTGTAC AGCCAGCATC 360 CTTAATCTCT GTGCCATCAG CATAGACAGGTACACTGCAG TGGTCATGCC CGTTCACTAC 420 CAGCATGGCA CGGGACAGAG CTCCTGTCGGCGCGTGGCCC TCATGATCAC GGCCGTCTGG 480 GTACTGGCCT TTGCTGTGTC CTGCCCTCTTCTGTTTGGCT TTAATACCAC AGGGGACCCC 540 ACTGTCTGCT CCATCTCCAA CCCTGATTTTGTCATCTACT CTTCAGTGGT GTCCTTCTAC 600 CTGCCCTTTG GAGTGACTGT CCTTGTCTATGCCAGAATCT ATGTGGTGCT GAAACAAAGG 660 AGACGGAAAA GGATCCTCAC TCGACAGAACAGTCAGTGCA ACAGTGTCAG GCCTGGCTTC 720 CCCCAACAAA CCCTCTCTCC TGACCCGGCACATCTGGAGC TGAAGCGTTA CTACAGCATC 780 TGCCAGGACA CTGCCTTGGG TGGACCAGGCTTCCAAGAAA GAGGAGGAGA GTTGAAAAGA 840 GAGGAGAAGA CTCGGAATTC CCTGAGTCCCACCATAGCGC CTAAGCTCAG CTTAGAAGTT 900 CGAAAGCTCA GCAATGGCAG ATTATCGACATCTTTGAAGC TGGGGCCCCT GCAACCTCGG 960 GGAGTGCCAC TTCGGGAGAA GAAGGCAACCCAAATGGTGG CCATTGTGCT TGGGGCCTTC 1020 ATTGTCTGCT GGCTGCCCTT CTTCTTGACCCATGTTCTCA ATACCCACTG CCAGACATGC 1080 CACGTGTCCC CAGAGCTTTA CAGTGCCACGACATGGCTGG GCTACGTGAA TAGCGCCCTC 1140 AACCCTGTGA TCTATACCAC CTTCAATATCGAGTTCCGGA AAGCCTTCCT CAAGATCCTG 1200 TCTTGCTGAG GGAGCAGAAG AGGGAACACTCTTTGTACCC ATTTCTAGCT GCCAGGCTGT 1260 T 1261

We claim:
 1. An isolated nucleic acid molecule comprising SEQ ID NO:1.2. An isolated nucleic acid molecule encoding SEQ ID NO:2.
 3. A probefor dopamine receptors comprising at least fourteen contiguousnucleotides of a nucleic acid molecule encoding a rat D₄ dopaminereceptor as shown in Sequence ID No. 1 between nucleotides 1 and 3263.4. The probe of claim 3 further comprising a label selected from thegroup consisting of dyes, radiolabels, tomography positron emissionlabels, magnetic resonance imaging labels, fluorescent labels, andenzymes.